Immune tolerance is central to the immune system's ability to differentiate between self and foreign proteins. Central tolerance is initially achieved during thymic selection by the deletion of self-reactive T cells. However, central tolerance is incomplete, and further immune regulation is required in the periphery. Peripheral mechanisms of T cell regulation include the induction of anergy, activation induced cell death, and regulatory T cells.
Within the CD4+ T lymphocyte cell population, three categories of regulatory T cells have been described: TH3 cells, Type 1 regulatory (Tr1) cells, and CD4+CD25+ T regulatory cells (“Treg”). TH3 cells function via the secretion of TGF-β and can be generated in vitro by stimulation in the presence of IL-4 or in vivo through oral administration of low dose antigens (Chen et al., Science 265:1237-1240, 1994; Inobe et al., Eur. J. Immunol. 28:2780-2790, 1998). Type 1 regulatory T cells (Tr1) suppress T cells through the production of IL-10 and TGF-β and are derived by stimulation of memory T cells in the presence of IL-10 (Groux et al., Nature 389:737-742, 1996; Groux et al., J. Exp. Med. 184:19-29, 1996). CD4+CD25+ regulatory T cells (Treg) are thought to function as a regulator of autoimmunity by suppressing the proliferation and/or cytokine production of CD4+CD25− T cell responder cells at the site of inflammation.
CD4+CD25+ Treg cells are known to be present in both humans and mice and are characterized by expression of CD25 (for review, see Sakaguchi et al., Immunol. Rev. 182:18-32). Treg cells isolated from human peripheral blood are highly differentiated memory cells based on their FACS staining characteristics and short telomere length and historically are thought to be derived from the thymus (Taams et al., Eur. J. Immunol. 32:1621-1630, 2002; Jonuleit et al., J. Exp. Med. 193:1285-1294, 2001). In humans, Tregs are believed to represent 1-3% of all CD4+ T cells and require activation to induce suppressor function. The suppressive function of these Treg cells is mediated via cell-cell contact and is abrogated by the addition of IL-2 (Baecher-Allan et al., J. Immunol 167:1245-1253, 2001).
The Treg population is reduced in autoimmune-prone animals and humans (see Salomon et al., Immunity 12:431-440, 2000; Kukreja et al., J. Clin. Invest. 109:131-140, 2002). Mice carrying the X-linked scurfy mutation develop a multi-organ autoimmune disease and lack conventional CD4+CD25+regulatory T cells (Fontenot et al., Nat. Immunol. 4:330-336, 2003; Khattri et al., Nat. Immunol. 4:337-342, 2003). It has been shown that the gene mutated in these mice is FoxP3 which encodes a member of the forkhead/winged helix family and acts as a transcriptional repressor (Schubert et al., J. Biol. Chem. 276:37672-37679, 2001). In mice, FoxP3 has been shown to be expressed exclusively in CD4+CD25+ Treg cells and is not induced upon activation of CD25− cells. However, when FoxP3 is introduced via retrovirus or via transgene expression, naïve CD4+CD25− T cells are converted to Treg cells (Hori et al., Science 299:1057-1061, 2003). In humans, it has been noted that mutations in FoxP3 lead to a severe lymphoproliferative disorder known as IPEX (immunodysregulation, polyendocrinopathy, enteropathy, X-linked) syndrome, characterized by lymphoproliferative disease, insulin-dependent diabetes, thyroiditis, eczema and death at an early age (see Wildin et al., J. Med. Genet. 39:537-545, 2002).
Due to their low frequency in peripheral blood, freshly isolated human CD4+CD25+ T cells with suppressive function are difficult to isolate and expand. In the autoimmune NOD mouse model, in which mice are transgenic for a single T cell receptor, one group of investigators has recently isolated naturally occurring antigen-specific Treg cells from mouse spleen and lymph nodes, expanded the cells and demonstrated that transfer of these cells to the diabetic prone NOD mouse can suppress the development of diabetes (Tang et al., J. Exp. Med. 199:1455-1465, 2004, Masteller et al., J. Immunol 175:3053-3059, 2005; Tarbell et al., J. Exp Med 199:1467-1477, 2004). This approach demonstrates the therapeutic benefit of Treg transfer to treat autoimmune disease. However, the approach used in the NOD mouse model is not therapeutically applicable to human subjects, due to the requirement that a large number of rare CD4+CD25+ T cells (approximately 4% of circulating T cells) be isolated from the peripheral blood. Further, this mouse model contains a single fixed T cell receptor (TCR) and does not address the problem of following TCR repertoire evolution or identifying antigen-specific T cells in complex systems where a polyclonal T cell response is present. Similar studies have not been possible in human subjects due to the low frequency of antigen-specific Treg cells circulating in the peripheral blood, especially with respect to autoreactive T cells.
Given the important role CD4+CD25+ regulatory T cells play in immune tolerance, there is a need to develop methods for generating, selecting and expanding human antigen-specific regulatory CD4+CD25+ T cells from the peripheral blood of a subject in need thereof for use in the treatment and/or prevention of autoimmune diseases, inflammatory conditions and for the prevention of graft rejection in a recipient following solid organ or stem cell transplantation.